Frederik Boernke wrote:
> Hi all,
>> I did some PCR mutagenesis on my gene using Stratagene's quick
> change kit. Everything seemed ok so far, since sequencing of the
> respective region confirmed the nucleotide exchange. However, when
> looking at the oligoes used I realized a mistake at the 3' end which
> actually should introduce a frame shift into my sequence.
> The last bases of the oligo are: GAT TTG G but my sequence is GAT TGG.
> The oligos where HPLC purified so they should be alright. Sequencing of
> several independent mutagenized clones revealed no frame shift, the
> additional T disappeared. How can this happen? Any ideas?
>
Hi Ricky,
Sounds like the proofreading activity of the Pfu polymerase did a good
job. I guess the 3´terminus of your primer did not anneal well because of
the mismatch in position -3. Pfu may have recognised that as an error and
removed 3 bases from the 3´end of the primer before polymerisation.
Frank